Method of treating dyslipidemic disorders

ABSTRACT

The invention provides methods of treating or preventing a condition or disorder associated with dyslipidemia with compositions comprising apolipoprotein-sphingomyelin complexes. The methods of the invention permit reduction, by 4- to 20-fold, of the amount of apolipoprotein required for therapeutic administration to bring about an ameliorative effect.

[0001] This application claims priority to U.S. Provisional ApplicationSerial Number 60/381,512, filed May 17, 2002, incorporated herein byreference in its entirety.

1. TECHNICAL FIELD

[0002] The invention provides methods of treating or preventing adisease, condition or disorder associated with dyslipidemia withapolipoprotein-sphingomyelin complexes or pharmaceutical compositionsthereof. The invention further provides compositions for treating orpreventing a disease, condition or disorder associated with dyslipidemiaand methods for the production of the compositions.

2. BACKGROUND OF THE INVENTION

[0003] Circulating cholesterol is carried by plasma lipoproteins—complexparticles of lipid and protein composition that transport lipids in theblood. Four major classes of lipoprotein particles circulate in plasmaand are involved in the fat-transport system: chylomicrons, very lowdensity lipoprotein (VLDL), low density lipoprotein (LDL) and highdensity lipoprotein (HDL). Chylomicrons constitute a short-lived productof intestinal fat absorption. VLDL and particularly, LDL, areresponsible for the delivery of cholesterol from the liver (where it issynthesized or obtained from dietary sources) to extrahepatic tissues,including the arterial walls. HDL, by contrast, mediates reversecholesterol transport (RCT), the removal of cholesterol fromextrahepatic tissues to the liver, where it is catabolized, eliminatedor recycled. HDL also plays a role in inflammation, transportingoxidized lipids and interleukin.

[0004] Lipoprotein particles have a hydrophobic core comprised ofcholesterol (normally in the form of a cholesteryl ester) andtriglycerides. The core is surrounded by a surface coat comprisingphospholipids, unesterified cholesterol and apolipoproteins.Apolipoproteins mediate lipid transport, and some may interact withenzymes involved in lipid metabolism. At least ten apolipoproteins havebeen identified, including: ApoA-I, ApoA-II, ApoA-IV, ApoA-V, ApoB,ApoC-I, ApoC-II, ApoC-III, ApoD, ApoE, ApoJ and ApoH. Other proteinssuch as LCAT (lecithin:cholesterol acyltransferase), CETP (cholesterylester transfer protein), PLTP (phospholipid transfer protein) and PON(paraoxonase) are also found associated with lipoproteins.

[0005] Cardiovascular diseases such as coronary heart disease, coronaryartery disease and atherosclerosis are linked overwhelmingly to elevatedserum cholesterol levels. For example, atherosclerosis is a slowlyprogressive disease characterized by the accumulation of cholesterolwithin the arterial wall. Compelling evidence supports the theory thatlipids deposited in atherosclerotic lesions are derived primarily fromplasma LDLs; thus, LDLs have popularly become known as “bad”cholesterol. In contrast, HDL serum levels correlate inversely withcoronary heart disease. Indeed, high serum levels of HDLs are regardedas a negative risk factor. It is hypothesized that high levels of plasmaHDLs are not only protective against coronary artery disease, but mayactually induce regression of atherosclerotic plaque (see, e.g., Badimonet al., 1992, Circulation 86(Suppl. III):86-94; Dansky and Fisher, 1999,Circulation 100:1762-63; Tangirala et al., 1999, Circulation100(17):1816-22; Fan et al., 1999, Atherosclerosis 147(1):139-45;Deckert et al., 1999, Circulation 100(11):1230-35; Boisvert et al.,1999, Arterioscler. Thromb. Vasc. Biol.19(3):525-30; Benoit et al.,1999, Circulation 99(1):105-10; Holvoet et al., 1998, J. Clin. Invest.102(2):379-85; Duverger et al., 1996, Circulation 94(4):713-17; Miyazakiet al., 1995, Arterioscler. Thromb. Vasc. Biol. 15(11):1882-88; Mezdouret al., 1995, Atherosclerosis 113(2):237-46; Liu et al., 1994, J. LipidRes. 35(12):2263-67; Plump et al., 1994, Proc. Nat. Acad. Sci. USA91(20):9607-11; Paszty et al., 1994, J. Clin. Invest. 94(2):899-903; Sheet al, 1992, Chin. Med. J. (Engl). 105(5):369-73; Rubin et al., 1991,Nature 353(6341):265-67; She et al., 1990, Ann. NY Acad. Sci.598:339-51; Ran, 1989, Chung Hua Ping Li Hsueh Tsa Chih (also translatedas: Zhonghua Bing Li Xue Za Zhi) 18(4):257-61; Quezado et al., 1995, J.Pharmacol. Exp. Ther. 272(2):604-11; Duverger et al., 1996,Arterioscler. Thromb. Vasc. Biol. 16(12):1424-29; Kopfler et al., 1994,Circulation; 90(3):1319-27; Miller et al., 1985, Nature314(6006):109-11; Ha et al., 1992, Biochim. Biophys. Acta1125(2):223-29; Beitz et al., 1992, Prostaglandins Leukot. Essent. FattyAcids 47(2):149-52). As a consequence, HDLs have popularly become knownas “good” cholesterol.

[0006] The “protective” role of HDL has been confirmed in a number ofstudies (e.g., Miller et al., 1977, Lancet 1(8019):965-68; Whayne etal., 1981, Atherosclerosis 39:411-19). In these studies, the elevatedlevels of LDL appear to be associated with increased cardiovascularrisk, whereas high HDL levels seem to confer cardiovascular protection.In vivo studies have further demonstrated the protective role of HDL,showing that HDL infusions into rabbits may hinder the development ofcholesterol induced arterial lesions (Badimon et al., 1989, Lab. Invest.60:455-61) and/or induce their regression (Badimon et al., 1990, J.Clin. Invest. 85:1234-41).

2.1. REVERSE CHOLESTEROL TRANSPORT, HDL AND APOLIPOPROTEIN A-I

[0007] The reverse cholesterol transport (RCT) pathway functions toeliminate cholesterol from most extrahepatic tissues and is crucial tomaintaining the structure and function of most cells in the body. RCTconsists mainly of three steps: (a) cholesterol efflux, i.e., theinitial removal of cholesterol from various pools of peripheral cells;(b) cholesterol esterification by the action of lecithin:cholesterolacyltransferase (LCAT), preventing a re-entry of effluxed cholesterolinto cells; and (c) uptake of HDL cholesterol and cholesteryl esters toliver cells for hydrolysis, then recycling, storage, excretion in bileor catabolism to bile acids.

[0008] LCAT, the key enzyme in RCT, is produced by the liver andcirculates in plasma associated with the HDL fraction. LCAT convertscell-derived cholesterol to cholesteryl esters, which are sequestered inHDL destined for removal (see Jonas 2000, Biochim. Biophys. Acta1529(1-3):245-56). Cholesteryl ester transfer protein (CETP) andphospholipid transfer protein (PLTP) contribute to further remodeling ofthe circulating HDL population. CETP moves cholesteryl esters made byLCAT to other lipoproteins, particularly ApoB-comprising lipoproteins,such as VLDL and LDL. PLTP supplies lecithin to HDL. HDL triglyceridesare catabolized by the extracellular hepatic triglyceride lipase, andlipoprotein cholesterol is removed by the liver via several mechanisms.

[0009] The functional characteristics of HDL particles are mainlydetermined by their major apolipoprotein components such as ApoA-I andApoA-II. Minor amounts of ApoC-I, ApoC-II, ApoC-III, ApoD, ApoA-IV,ApoE, ApoJ have also been observed associated with HDL. HDL exists in awide variety of different sizes and different mixtures of theabove-mentioned constituents, depending on the status of remodelingduring the metabolic RCT cascade or pathway.

[0010] Each HDL particle usually comprises at least one molecule, andusually two to four molecules, of ApoA-I. HDL particles may alsocomprise only ApoE (γ-LpE particles), which are known to also beresponsible for cholesterol efflux, as described by Prof. Gerd Assmann(see, e.g., von Eckardstein et al., 1994, Curr Opin Lipidol.5(6):404-16). ApoA-I is synthesized by the liver and small intestine aspreproapolipoprotein A-I, which is secreted as proapolipoprotein A-I(proApoA-I) and rapidly cleaved to generate the plasma form of ApoA-I, asingle polypeptide chain of 243 amino acids (Brewer et al., 1978,Biochem. Biophys. Res. Commun. 80:623-30). PreproApoA-I that is injectedexperimentally directly into the bloodstream is also cleaved into theplasma form of ApoA-I (Klon et al., 2000, Biophys. J. 79(3):1679-85;Segrest et al., 2000, Curr. Opin. Lipidol. 11(2):105-15; Segrest et al.,1999, J. Biol. Chem. 274 (45):31755-58).

[0011] ApoA-I comprises 6 to 8 different 22-amino acid a-helices orfunctional repeats spaced by a linker moiety that is frequently proline.The repeat units exist in amphipathic helical conformation (Segrest etal., 1974, FEBS Lett. 38: 247-53) and confer the main biologicalactivities of ApoA-I, i.e., lipid binding and lecithin cholesterol acyltransferase (LCAT) activation.

[0012] ApoA-I forms three types of stable complexes with lipids: small,lipid-poor complexes referred to as pre-β-1 HDL; flattened discoidalparticles comprising polar lipids (phospholipid and cholesterol)referred to as pre-β-2 HDL; and spherical particles, comprising bothpolar and nonpolar lipids, referred to as spherical or mature HDL(HDL_(L3) and HDL_(L2)). Most HDL in the circulating population compriseboth ApoA-I and ApoA-II (the “AI/AII-HDL fraction”). However, thefraction of HDL comprising only ApoA-I (the “AI-HDL fraction”) appearsto be more effective in RCT. Certain epidemiologic studies support thehypothesis that the Apo-AI-HDL fraction is anti-atherogenic. (Parra etal., 1992, Arterioscler. Thromb. 12:701-07; Decossin et al., 1997, Eur.J. Clin. Invest. 27:299-307).

[0013] Although the mechanism for cholesterol transfer from the cellsurface (i.e., cholesterol efflux) is unknown, it is believed that thelipid-poor complex, pre-β-1 HDL, is the preferred acceptor forcholesterol transferred from peripheral tissue involved in RCT. (SeeDavidson et al., 1994, J. Biol. Chem. 269:22975-82; Bielicki et al.,1992, J. Lipid Res. 33:1699-1709; Rothblat et al., 1992, J. Lipid Res.33:1091-97; and Kawano et al., 1993, Biochemistry 32:5025-28; Kawano etal., 1997, Biochemistry 36:9816-25). During this process of cholesterolrecruitment from the cell surface, pre-β-1 HDL is rapidly converted topre-β-2 HDL. PLTP may increase the rate of pre-β-2 HDL disc formation,but data indicating a role for PLTP in RCT is lacking. LCAT reactspreferentially with discoidal, small (pre-β) and spherical (i.e.,mature) HDL, transferring the 2-acyl group of lecithin or otherphospholipids to the free hydroxyl residue of cholesterol to generatecholesteryl esters (retained in the HDL) and lysolecithin. The LCATreaction requires ApoA-I as an activator; i.e., ApoA-I is the naturalcofactor for LCAT. The conversion of cholesterol sequestered in the HDLto its ester prevents re-entry of cholesterol into the cell, the netresult being that cholesterol is removed from the cell.

[0014] Cholesteryl esters in the mature HDL particles in the ApoAI-HDLfraction (i.e., comprising ApoA-I and no ApoA-II) are removed by theliver and processed into bile more effectively than those derived fromHDL comprising both ApoA-I and ApoA-II (the Al/AII-HDL fraction). Thismay be owing, in part, to the more effective binding of ApoAI-HDL to thehepatocyte membrane. The existence of an HDL receptor has beenhypothesized, and a scavenger receptor, class B, type I (SR—BI) has beenidentified as an HDL receptor (Acton et al., 1996, Science 271:518-20;Xu et al., 1997, Lipid Res. 38:1289-98). SR—BI is expressed mostabundantly in steroidogenic tissues (e.g., the adrenals), and in theliver (Landschulz et al., 1996, J. Clin. Invest. 98:984-95; Rigotti etal., 1996, J. Biol. Chem. 271:33545-49).

[0015] CETP may also play a role in RCT. Changes in CETP activity or itsacceptors, VLDL and LDL, play a role in “remodeling” the HDL population.For example, in the absence of CETP, the HDLs become enlarged particlesthat are not cleared. (For reviews of RCT and HDLs, see Fielding andFielding, 1995, J. Lipid Res. 36:211-28; Barrans et al., 1996, Biochem.Biophys. Acta 1300:73-85; Hirano et al., 1997, Arterioscler. Thromb.Vasc. Biol. 17(6):1053-59).

[0016] HDL also plays a role in the reverse transport of other lipidsand in detoxification, i.e., the transport of lipids from cells, organs,and tissues to the liver for catabolism and excretion. Such lipidsinclude sphingomyelin (SM), oxidized lipids, and lysophophatidylcholine.For example, Robins and Fasulo (1997, J. Clin. Invest. 99:380-84) haveshown that HDLs stimulate the transport of plant sterol by the liverinto bile secretions.

[0017] The major component of HDL, ApoA-I, can associate with SM invitro. When ApoA-I is reconstituted in vitro with bovine brain SM(BBSM), a maximum rate of reconstitution occurs at 28° C., thetemperature approximating the phase transition temperature for BBSM(Swaney, 1983, J. Biol. Chem. 258(2), 1254-59). At BBSM:ApoA-I ratios of7.5:1 or less (wt/wt), a single reconstituted homogeneous HDL particleis formed that comprises three ApoA-I molecules per particle and thathas a BBSM:ApoA-I molar ratio of 360:1. It appears in the electronmicroscope as a discoidal complex similar to that obtained byrecombination of ApoA-I with phosphatidylcholine at elevated ratios ofphospholipid/protein. At BBSM:ApoA-I ratios of 15:1 (wt/wt), however,larger-diameter discoidal complexes form that have a higherphospholipid:protein molar ratio (535:1). These complexes aresignificantly larger, more stable, and more resistant to denaturationthan ApoA-I complexes formed with phosphatidylcholine.

[0018] Sphingomyelin (SM) is elevated in early cholesterol acceptors(pre-β-HDL and γ-migrating ApoE-comprising lipoprotein), suggesting thatSM might enhance the ability of these particles to promote cholesterolefflux (Dass and Jessup 2000, J. Pharm. Pharmacol. 52:731-61; Huang etal., 1994, Proc. Natl. Acad. Sci. USA 91:1834-38; Fielding and Fielding1995, J. Lipid Res. 36:211-28).

2.2. PROTECTIVE MECHANISM OF HDL AND APOA-I

[0019] Recent studies of the protective mechanism(s) of HDL have focusedon apolipoprotein A-I (ApoA-I), the major component of HDL. High plasmalevels of ApoA-I are associated with absence or reduction of coronarylesions (Maciejko et al., 1983, N. Engl. J. Med. 309:385-89; Sedlis etal., 1986, Circulation 73:978-84).

[0020] The infusion of ApoA-I or of HDL in experimental animals exertssignificant biochemical changes, as well as reduces the extent andseverity of atherosclerotic lesions. After an initial report by Maciejkoand Mao (1982, Arteriosclerosis 2:407a), Badimon et al., (1989, Lab.Invest. 60:455-61; 1989, J. Clin. Invest. 85:1234-41) found that theycould significantly reduce the extent of atherosclerotic lesions(reduction of 45%) and their cholesterol ester content (reduction of58.5%) in cholesterol-fed rabbits, by infusing HDL (d=1.063-1.325 g/ml).They also found that the infusions of HDL led to a close to a 50%regression of established lesions. Esper et al. (1987, Arteriosclerosis7:523a) have shown that infusions of HDL can markedly change the plasmalipoprotein composition of Watanabe rabbits with inheritedhypercholesterolemia, which develop early arterial lesions. In theserabbits, HDL infisions can more than double the ratio between theprotective HDL and the atherogenic LDL.

[0021] The potential of HDL to prevent arterial disease in animal modelshas been further underscored by the observation that ApoA-I can exert afibrinolytic activity in vitro (Saku et al., 1985, Thromb. Res. 39:1-8).Ronneberger (1987, Xth Int. Congr. Pharmacol., Sydney, 990) demonstratedthat ApoA-I can increase fibrinolysis in beagle dogs and in Cynomologousmonkeys. A similar activity can be noted in vitro on human plasma.Ronneberger was able to confirm a reduction of lipid deposition andarterial plaque formation in ApoA-I treated animals.

[0022] In vitro studies indicate that complexes of ApoA-I and lecithincan promote the efflux of free cholesterol from cultured arterial smoothmuscle cells (Stein et al., 1975, Biochem. Biophys. Acta, 380:106-18).By this mechanism, HDL can also reduce the proliferation of these cells(Yoshida et al., 1984, Exp. Mol Pathol. 41:258-66).

[0023] Two naturally occurring human mutations of ApoA-I have beenisolated in which an arginine residue is mutated to cysteine. Inapolipoprotein A-I_(Milano) (ApoA-I_(M)), this substitution occurs atresidue 173, whereas in apolipoprotein A-I_(Paris) (ApoA-I_(P)), thissubstitution occurs at residue 151 (Franceschini et al., 1980, J. Clin.Invest. 66:892-900; Weisgraber et al., 1983, J. Biol. Chem. 258:2508-13;Bruckert et al., 1997, Atherosclerosis 128:121-28; Daum et al., 1999, J.Mol. Med. 77:614-22; Klon et al., 2000, Biophys. J. 79(3):1679-85).

[0024] Reconstituted HDL particles comprising disulfide-linkedhomodimers of either ApoA-I_(M) or ApoA-I_(P) are similar toreconstituted HDL particles comprising wild-type ApoA-I in their abilityto clear dimyristoylphosphatidylcholine (DMPC) emulsions and theirability to promote cholesterol efflux (Calabresi et al., 1997b,Biochemistry 36:12428-33; Franceschini et al., 1999, Arterioscler.Thromb. Vasc. Biol. 19:1257-62; Daum et al., 1999, J. Mol. Med.77:614-22). In both mutations, heterozygous individuals have decreasedlevels of HDL but paradoxically, are at a reduced risk foratherosclerosis (Franceschini et al., 1980, J. Clin. Invest. 66:892-900;Weisgraber et al., 1983, J. Biol. Chem. 258:2508-13; Bruckert et al.,1997, Atherosclerosis 128:121-28). Reconstituted HDL particlescomprising either variant are capable of LCAT activation, although withdecreased efficiency when compared with reconstituted HDL particlescomprising wild-type ApoA-I (Calabresi et al., 1997a, Biochem. Biophys.Res. Commun. 232:345-49; Daum et al., 1999, J. Mol. Med. 77:614-22).

[0025] The ApoA-I_(M) mutation is transmitted as an autosomal dominanttrait; 8 generations of carriers within a family have been identified(Gualandri et al., 1984, Am. J. Hum. Genet. 37:1083-97). The status ofan ApoA-I_(M) carrier individual is characterized by a remarkablereduction in HDL-cholesterol level. In spite of this, carrierindividuals do not apparently show any increased risk of arterialdisease. Indeed, by examination of genealogical records, it appears thatthese subjects may be “protected” from atherosclerosis (Sirtori et al.,2001, Circulation, 103: 1949-1954; Roma et al., 1993, J. Clin. Invest.91(4):1445-520).

[0026] The mechanism of the possible protective effect of ApoA-I_(M) incarriers of the mutation seems to be linked to a modification in thestructure of the mutant ApoA-I_(M), with loss of one alpha-helix and anincreased exposure of hydrophobic residues (Franceschini et al., 1985,J. Biol. Chem. 260:1632-35). The loss of the tight structure of themultiple alpha-helices leads to an increased flexibility of themolecule, which associates more readily with lipids, compared to normalApoA-I. Moreover, apolipoprotein-lipid complexes are more susceptible todenaturation, thus suggesting that lipid delivery is also improved inthe case of the mutant.

[0027] Bielicki, et al. (1997, Arterioscler. Thromb. Vasc. Biol. 17(9):1637-43) has demonstrated that ApoA-I_(M) has a limited capacity torecruit membrane cholesterol compared with wild-type ApoA-I. Inaddition, nascent HDL formed by the association of ApoA-I_(M) withmembrane lipids was predominantly 7.4-nm particles rather than larger 9-and 11-nm complexes formed by wild-type ApoA-I. These observationsindicate that the Arg₁₇₃→Cys₁₇₃ substitution in the ApoA-I primarysequence interfered with the normal process of cellular cholesterolrecruitment and nascent HDL assembly. The mutation is apparentlyassociated with a decreased efficiency for cholesterol removal fromcells. Its antiatherogenic properties may therefore be unrelated to RCT.

[0028] The most striking structural change attributed to theArg₁₇₃→Cys₁₇₃ substitution is the dimerization of ApoA-I_(M) (Bielickiet al., 1997, Arterioscler. Thromb. Vasc. Biol. 17 (9):1637-43).ApoA-I_(M) can form homodimers with itself and heterodimers withApoA-II. Studies of blood fractions comprising a mixture ofapolipoproteins indicate that the presence of dimers and complexes inthe circulation may be responsible for an increased eliminationhalf-life of apolipoproteins. Such an increased elimination half-lifehas been observed in clinical studies of carriers of the mutation (Gregget al., 1988, NATO ARW on Human Apolipoprotein Mutants: From GeneStructure to Phenotypic Expression, Limone S G). Other studies indicatethat ApoA-I_(M) dimers (ApoA-I_(M)/ApoA-I_(M)) act as an inhibitingfactor in the interconversion of HDL particles in vitro (Franceschini etal., 1990, J. Biol. Chem. 265:12224-31).

2.3. CURRENT TREATMENTS FOR DYSLIPIDEMIC AND RELATED DISORDERS

[0029] Dyslipidemic disorders are diseases associated with elevatedserum cholesterol and triglyceride levels and lowered serum HDL:LDLratios, and include hyperlipidemia, especially hypercholesterolemia,coronary heart disease, coronary artery disease, vascular andperivascular diseases, and cardiovascular diseases such asatherosclerosis. Syndromes associated with atherosclerosis such asintermittent claudication, caused by arterial insufficiency, are alsoincluded. A number of treatments are currently available for loweringthe elevated serum cholesterol and triglycerides associated withdyslipidemic disorders. However, each has its own drawbacks andlimitations in terms of efficacy, side-effects and qualifying patientpopulation.

[0030] Bile-acid-binding resins are a class of drugs that interrupt therecycling of bile acids from the intestine to the liver; e.g.,cholestyramine (Questran Light®, Bristol-Myers Squibb), and colestipolhydrochloride (Colestid®, The Upjohn Company). When taken orally, thesepositively-charged resins bind to the negatively charged bile acids inthe intestine. Because the resins cannot be absorbed from the intestine,they are excreted carrying the bile acids with them. The use of suchresins at best, however, only lowers serum cholesterol levels by about20%, and is associated with gastrointestinal side-effects, includingconstipation and certain vitamin deficiencies. Moreover, since theresins bind other drugs, other oral medications must be taken at leastone hour before or four to six hours subsequent to ingestion of theresin; thus, complicating heart patient's drug regimens.

[0031] Statins are cholesterol lowering agents that block cholesterolsynthesis by inhibiting HMGCoA reductase—the key enzyme involved in thecholesterol biosynthetic pathway. Statins, e.g., lovastatin (Mevacor®),simvastatin (Zocor®), pravastatin (Pravachol®), fluvastatin (Lescol®)and atorvastatin (Lipitor®), are sometimes used in combination withbile-acid-binding resins. Statins significantly reduce serum cholesteroland LDL-serum levels, and slow progression of coronary atherosclerosis.However, serum HDL cholesterol levels are only moderately increased. Themechanism of the LDL lowering effect may involve both reduction of VLDLconcentration and induction of cellular expression of LDL-receptor,leading to reduced production and/or increased catabolism of LDLs. Sideeffects, including liver and kidney dysfunction are associated with theuse of these drugs (The Physicians Desk Reference (56^(th) ed., 2002)Medical Economics).

[0032] Niacin (nicotinic acid) is a water soluble vitamin B-complex usedas a dietary supplement and antihyperlipidemic agent. Niacin diminishesproduction of VLDL and is effective at lowering LDL. In some cases, itis used in combination with bile-acid binding resins. Niacin canincrease HDL when used at adequate doses, however, its usefulness islimited by serious side effects when used at such high doses. Niaspan®is a form of extended-release niacin that produces fewer side effectsthan pure niacin. Niacin/Lovastatin (Nicostatin®) is a formulationcontaining both niacin and lovastatin and combines the benefits of eachdrug.

[0033] Fibrates are a class of lipid-lowering drugs used to treatvarious forms of hyperlipidemia (i.e., elevated serum triglycerides)that may also be associated with hypercholesterolemia. Fibrates appearto reduce the VLDL fraction and modestly increase HDL—however theeffects of these drugs on serum cholesterol is variable. In the UnitedStates, fibrates such as clofibrate (Atromid-S®), fenofibrate (Tricor®)and bezafibrate (Bezalip®) have been approved for use as antilipidemicdrugs, but have not received approval as hypercholesterolemia agents.For example, clofibrate is an antilipidemic agent that acts (via anunknown mechanism) to lower serum triglycerides by reducing the VLDLfraction. Although serum cholesterol may be reduced in certain patientsubpopulations, the biochemical response to the drug is variable, and isnot always possible to predict which patients will obtain favorableresults. Atromid-S® has not been shown to be effective for prevention ofcoronary heart disease. The chemically and pharmacologically relateddrug, gemfibrozil (Lopid®) is a lipid regulating agent that moderatelydecreases serum triglycerides and VLDL cholesterol, and moderatelyincreases HDL cholesterol—the HDL₂ and HDL₃ subfractions as well as bothApoA-I and A-II (i.e., the AI/AMT-HDL fraction). However, the lipidresponse is heterogeneous, especially among different patientpopulations. Moreover, while prevention of coronary heart disease wasobserved in male patients between 40-55 without history or symptoms ofexisting coronary heart disease, it is not clear to what extent thesefindings can be extrapolated to other patient populations (e.g., women,older and younger males). Indeed, no efficacy was observed in patientswith established coronary heart disease. Serious side-effects areassociated with the use of fibrates including toxicity such asmalignancy, (especially gastrointestinal cancer), gallbladder diseaseand an increased incidence in non-coronary mortality. These drugs arenot indicated for the treatment of patients with high LDL or low HDL astheir only lipid abnormality (The Physicians Desk Reference (56^(th)ed., 2002) Medical Economics). Oral estrogen replacement therapy may beconsidered for moderate hypercholesterolemia in post-menopausal women.However, increases in HDL may be accompanied with an increase intriglycerides. Estrogen treatment is, of course, limited to a specificpatient population (postmenopausal women) and is associated with seriousside effects including induction of malignant neoplasms, gall bladderdisease, thromboembolic disease, hepatic adenoma, elevated bloodpressure, glucose intolerance, and hypercalcemia.

[0034] The need therefore exists for safer drugs that are moreefficacious in lowering serum cholesterol, increasing HDL serum levels,preventing and/or treating diseases, conditions or disorders associatedwith dyslipidemia.

[0035] For example, HDL, as well as recombinant forms of ApoA-Icomplexed with phospholipids can serve as sinks/scavengers for apolar oramphipathic molecules, e.g., cholesterol and derivatives (oxysterols,oxidized sterols, plant sterols, etc.), cholesterol esters,phospholipids and derivatives (oxidized phospholipids), triglycerides,oxidation products, and lipopolysaccharides (LPS) (see, e.g., Casas etal., 1995, J. Surg. Res. Nov;59(5):544-52). HDL can also serve as also ascavenger for TNF-α and other lymphokines. HDL can also serve as acarrier for human serum paraoxonases, e.g., PON-1,-2,-3. Paraoxonase, anesterase associated with HDL, is important for protecting cellcomponents against oxidation. Oxidation of LDL, which occurs duringoxidative stress, appears directly linked to development ofatherosclerosis (Aviram, 2000, Free Radic. Res. 33 Suppl:S85-97).Paraoxonase appears to play a role in susceptibility to atherosclerosisand cardiovascular disease (Aviram, 1999, Mol. Med. Today 5(9):381-86).Human serum paraoxonase (PON-1) is bound to high-density lipoproteins(HDLs). Its activity is inversely related to atherosclerosis. PON-1hydrolyzes organophosphates and may protect against atherosclerosis byinhibition of the oxidation of HDL and low-density lipoprotein (LDL)(Aviram, 1999, Mol. Med. Today 5(9):381-86). Experimental studiessuggest that this protection is associated with the ability of PON-1 tohydrolyze specific lipid peroxides in oxidized lipoproteins.Interventions that preserve or enhance PON-1 activity may help to delaythe onset of atherosclerosis and coronary heart disease.

[0036] HDL further has a role as an antithrombotic agent and fibrinogenreducer, and as an agent in hemorrhagic shock (Cockerill et al., WO01/13939, published March 1, 2001). HDL, and ApoA-I in particular, hasbeen show to facilitate an exchange of lipopolysaccharide produced bysepsis into lipid particles comprising ApoA-I, resulting in thefunctional neutralization of the lipopolysaccharide (Wright et al.,WO9534289, published December 21, 1995; Wright et al., U.S. Pat. No.5,928,624 issued July 27, 1999; Wright et al., U.S. Pat. No. 5,932,536,issued Aug. 3, 1999).

[0037] The therapeutic use of ApoA-I, ApoA-I_(Milano), ApoA-I_(Paris)and other variants, as well as reconstituted HDL, is presently limited,however, by the large amount of apolipoprotein required for therapeuticadministration and by the cost of protein production, considering thelow overall yield of production. It has been suggested by early clinicaltrials that the dose range is between 1.5-4 g of protein per infusionfor treatment of cardiovascular diseases. The number of infusionsrequired for a full treatment is unknown. (See, e.g., Eriksson et al.,1999, Circulation 100(6):594-98; Carlson, 1995, Nutr. Metab. Cardiovasc.Dis. 5:85-91; Nanjee et al., 2000, Arterioscler. Thromb. Vasc. Biol.20(9):2148-55; Nanjee et al., 1999, Arterioscler. Thromb. Vasc. Biol.19(4):979-89; Nanjee et al., 1996, Arterioscler. Thromb. Vasc. Biol.16(9):1203-14). Thus, there is a need to develop new methods oftreatment of dyslipidemic diseases, conditions or disorders thatminimize the amount of apolipoprotein required for administration.

[0038] Citation or identification of any reference in Section 2 or inany other section of this application shall not be construed as anadmission that such reference is available as prior art to the presentinvention.

3. SUMMARY OF THE INVENTION

[0039] The invention provides methods of treating or preventing adisease, condition or disorder associated with dyslipidemia, whichmethods utilize apolipoprotein-sphingomyelin complexes or compositionscomprising apolipoprotein-sphingomyelin complexes, e.g.,apolipoproteinA-I-SM (ApoA-I-SM).

[0040] Quite surprisingly, it has been discovered that whenapolipoproteins such as ApoA-I are administered in the form ofapolipoprotein-sphingomyelin (“Apo-SM”) complexes, far lessapolipoprotein is required to achieve the same or better cholesterolmobilization and, hence, therapeutic benefit, than that provided byother apolipoprotein-lipid complexes. For example, whereas soybeanphosphatidylcholine (“soybeanPC”) treatment regimens requireadministration of from 20-50 mg/kg (or 1-4 g/person) apolipoproteinevery 2-5 days (i.v.), treatment regimens according to the inventionrequire the administration of only 0.05 to 25 mg/kg (40 mg to 2 g perperson) of apolipoprotein every 2-10 days (i.v.). Thus, the methods ofthe invention reduce by 2- to 25-fold the amount of apolipoproteinrequired for therapeutic benefit, thereby reducing substantially thecost of treatment, making the treatment regimen more convenient for thepatient and perhaps reducing possible adverse effects associated withadministration of the drug.

[0041] It has further been discovered that the mobilization ofcholesterol (elevation of HDL-cholesterol above a baseline level beforeadministration, wherein the baseline level is an initial level ofHDL-cholesterol, or is a level known to one of skill in the art as alevel that the patient in question would have, or an individual of thesize and gender of the would have, prior to administration of a drug) issignificantly sustained for a longer period of time for proApoA-I-SMcomplexes, i.e., longer than that of conventionalapolipoprotein-phospholipid complexes. Thus, treatments according to theinvention may be less frequent than current treatment protocols,typically about every 2 to 10 days, as compared with about every 2 to 5days, without loss of therapeutic benefit. Furthermore, a decreased dosemay be administered with the same frequency. In many embodiments,administration is about every 5 to 10 days, significantly reducing thenumber of clinic or hospital visits required by the patient.

[0042] The invention encompasses, in one embodiment, a pharmaceuticallyacceptable and injectable unit dosage which comprises less than 3500 mgof an apolipoprotein-sphingomyelin complex. In alternative embodiments,the composition comprises less than 1750 mg, less than 1400 mg, lessthan 700 mg and less than 350 mg per unit dosage form.

[0043] Use of the ApoA-I-SM complexes according to the invention is alsoadvantageous because lower anticipated doses of the volume of ApoA-I-SMcomplexes infused or injected leads to faster and easier administrationand improved patient comfort.

[0044] Use of the ApoA-I-SM complexes according to the invention isfurther advantageous because SM is a much more chemically stable lipidthan soybean phosphatidylcholine (soybean PC), hence there is greaterstability of ApoA-I complexes and longer product shelf-life compared toconvention complexes.

[0045] The methods of the invention provide benefit in virtually anycontext in which apolipoprotein therapy is advantageous. For example,the methods of the invention may be advantageously used to treat orprevent virtually any disease, condition or disorder associated withdyslipidemia that is treatable with apolipoproteins. Using the methodsof the invention, a dosage of apolipoprotein from 2- to 25-fold lessthan the effective dosage of apolipoprotein alone, or apolipoprotein andsoybean PC, may be administered. Since Apo-SM complexes are administeredsystemically, they can be used to treat or prevent atherosclerosis andstenosis, mobilizing cholesterol from a patient's entire vasculatureincluding small vessels.

4. DESCRIPTION OF THE FIGURES

[0046]FIG. 1 shows absolute changes in levels of HDL fraction ofunesterified cholesterol following administration of 15 mg/kg doses ofr-proApoA-I-SM and r-proApoA-I-POPC complexes. X-axis: time (hours).Y-axis: Change in free HDL cholesterol (mg/dL).

5. DETAILED DESCRIPTION OF THE INVENTION

[0047] The invention provides methods of treating or preventing adisease, condition or disorder associated with dyslipidemia utilizingapolipoprotein-sphingomyelin complexes or pharmaceutical compositionscomprising apolipoprotein-sphingomyelin (“Apo-SM”) complexes, e.g.,apolipoproteinA-I-SM (ApoA-I-SM).

[0048] As used herein, the terms “dyslipidemia” or “dyslipidemic” referto an abnormally elevated or decreased level of lipid in the bloodplasma, including, but not limited to, the altered level of lipidassociated with the following conditions: coronary heart disease;coronary artery disease; cardiovascular disease, hypertension,restenosis, vascular or perivascular diseases; dyslipidemic disorders;dyslipoproteinemia; high levels of low density lipoprotein cholesterol;high levels of very low density lipoprotein cholesterol; low levels ofhigh density lipoproteins; high levels of lipoprotein Lp(a) cholesterol;high levels of apolipoprotein B; atherosclerosis (including treatmentand prevention of atherosclerosis); hyperlipidemia;hypercholesterolemia; familial hypercholesterolemia (FH); familialcombined hyperlipidemia (FCH); lipoprotein lipase deficiencies, such ashypertriglyceridemia, hypoalphalipoproteinemia, andhypercholesterolemialipoprotein. Quite surprisingly, it has beendiscovered that when apolipoproteins such as Apo-AI are administered inthe form of apolipoprotein-sphingomyelin (Apo-SM) complexes, far lessapolipoprotein is required to achieve the same or better cholesterolmobilization and, hence, therapeutic benefit, than that provided byapolipoprotein-lipid complexes of phosphatidylcholines such assoybeanPC.

[0049] The invention encompasses the treatment of diseases or disordersassociated with dyslipidemia in a human in need thereof which comprisesadministering a therapeutically effective amount of anapolipoprotein-sphingomyelin complex to said human. Preferably thecomplex is a solid, including a lyophilized solid, suitable forreconstitution into a solution of apolipoprotein-sphingomyelin discoidalparticles. The administration is preferably parenteral, especiallyintravenous, bolus injection, intramuscular, subcutaneous and the like.Thus, the methods of the invention use 2- to 25-fold less apolipoproteinthan is required when a complex of apolipoprotein and soybean PC isadministered.

[0050] It has further been discovered that the mobilization ofcholesterol (elevation of HDL-cholesterol above the baseline (initial)level before administration) is sustained for a longer period of timefor proApoA-I-SM complexes than that ofapolipoprotein-phosphatidylcholine complexes. Thus, treatments accordingto the invention may be applied less frequently, typically about every 2to 10 days, without loss of therapeutic benefit. Furthermore, adecreased dose may be administered with the same frequency. In manyembodiments, treatment may be applied every 5 to 10 days, significantlyreducing the number of clinic or hospital visits required by thepatient.

[0051] Use of the ApoA-I-SM complexes according to the invention is alsoadvantageous because lower anticipated doses of the volume of ApoA-I-SMcomplexes infused or injected leads to faster and easier administrationand improved patient comfort.

[0052] Use of the ApoA-I-SM complexes according to the invention isfurther advantageous because SM is a much more chemically stable lipidthan soybean phosphatidylcholine (soybean PC), hence there is greaterstability of ApoA-I complexes and longer product shelf-life compared toconvention complexes.

[0053] The administration methods of the invention provide benefit invirtually any context in which apolipoprotein therapy is advantageous.For example, the methods of the invention may be advantageously used totreat or prevent virtually any disease, condition or disorder associatedwith dyslipidemia, or symptom thereof, responsive to apolipoproteins.Using the methods of the invention, a dosage of apolipoprotein from 2-to 25-fold less than the effective dosage currently known in the artwould be expected to be efficacious in treating or preventing thedisease or in bringing about an ameliorative effect. Since Apo-SMcomplexes are administered systemically, they can be used to treat orprevent atherosclerosis and stenosis, mobilizing cholesterol from apatient's entire vasculature including small vessels.

[0054] The invention is illustrated by working examples that demonstratethat the ApoA-I-SM complexes of the invention can increase theconcentration of HDL and increase cellular cholesterol efflux. Use ofthe ApoA-I-SM complexes disclosed herein in vivo in animal modelsresults in an increase of HDL cholesterol levels in plasma, which is anindicative of cholesterol mobilization/efflux by recombinant HDLparticles (i.e., ApoAI-SM-complexes). In addition, the increase of HDLcholesterol concentration induced by injection of ApoAI-SM particles issignificantly greater than that induced by ApoAI-phosphatidylcholineparticles (such as ApoAI-SoybeanPC and ApoAI-POPC).

[0055] For clarity of disclosure, and not by way of limitation, thedetailed description of the invention is divided into the subsectionsset forth below.

5.1. APOLIPOPROTEINS AND APOLIPOPROTEIN PEPTIDES

[0056] The invention utilizes apolipoprotein compositions in which anapolipoprotein is complexed with sphingomyelin (“Apo-SM complexes”).

[0057] Virtually any apolipoprotein or apolipoprotein derivative oranalogue that provides therapeutic benefit when used to treat or preventthe above-listed disorders may be complexed with SM and advantageouslyadministered at lower than conventional doses according to theinvention. Further, any protein or peptide of an a-helical nature thatalso activates LCAT may be used. Suitable apolipoproteins include, butare not limited to, preproapolipoprotein forms of ApoA-I, ApoA-II,ApoA-IV, ApoA-V and ApoE; pro- and mature forms of human ApoA-I,ApoA-II, ApoA-IV, and ApoE; and active polymorphic forms, isoforms,variants and mutants as well as truncated forms, the most common ofwhich are ApoA-I_(Milano) (ApoA-I_(M)) and ApoA-I_(Paris) (ApoA-I_(P)).Homo- and heterodimers (where feasible) of pro- and mature ApoA-I(Duverger et al., 1996, Arterioscler. Thromb. Vasc. Biol.16(12):1424-29), ApoA-I_(M) (Franceschini et al., 1985, J. Biol. Chem.260:1632-35), ApoA-I_(Paris) (Daum et al., 1999, J. Mol. Med.77:614-22), ApoA-II (Shelness et al., 1985, J. Biol. Chem.260(14):8637-46; Shelness et al., 1984, J. Biol. Chem. 259(15):9929-35),ApoA-IV (Duverger et al., 1991, Euro. J. Biochem. 201(2):373-83), ApoE(McLean et al., 1983, J. Biol. Chem. 258(14):8993-9000), ApoJ and ApoHmay also be utilized within the scope of the invention. Apolipoproteinsutilized by the invention also include recombinant or purifiedapolipoproteins. Apolipoprotein-SM complexes that may be used accordingto the methods of the invention include those disclosed in U.S. Pat. No.6,287,590, issued Sep. 11, 2001, which is incorporated herein byreference in its entirety.

[0058] Methods for obtaining apolipoproteins utilized by the inventionare well-known in the art, see, e.g., Chung et al., 1980, J. Lipid Res.21(3):284-91; Cheung et al., 1987, J. Lipid Res. 28(8):913-29.

[0059] Apolipoproteins utilized by the invention further includepeptides that correspond to apolipoproteins as well as agonists thatmimic the activity of ApoA-I, ApoA-I_(M), ApoA-II, ApoA-IV, and ApoE,such as those disclosed in U.S. Pat. Nos. 6,004,925, 6,037,323 and6,046,166 (issued to Dasseux et al.), and in U.S. Pat. No. 5,840,688(issued Nov. 24, 1998 to Tso); and which are incorporated herein byreference in their entireties.

[0060] Such peptides can be synthesized or manufactured using anytechnique for peptide synthesis known in the art, see, e.g., thetechniques described in U.S. Pat. Nos. 6,004,925, 6,037,323 and6,046,166. Stable preparations that have a long shelf life may be madeby lyophilizing the peptides—either to prepare bulk for reformulation,or to prepare individual aliquots or dosage units that can bereconstituted by rehydration with sterile water or an appropriatesterile buffered solution prior to administration to a subject.

[0061] The Apo-SM complexes may include a single type of apolipoprotein,or mixtures of two or more different apolipoproteins, which may bederived from the same or different species. Although not required, theApo-SM complexes will preferably comprise apolipoproteins derived fromthe animal species being treated, in order to avoid inducing an immuneresponse to the therapy.

[0062] The apolipoprotein(s) may be complexed with many types of SManalogues or derivatives. In general, for a SM analogue or derivative tobe effective in an Apo-SM complex of the invention, it can be imperviousto hydrolysis by LCAT, as is naturally occurring SM. SM is aphospholipid very similar in structure to phosphatidylcholine, but withan amide bond instead of an ester bond. SM is not a substrate for LCATand generally cannot be hydrolyzed by it. It can act, however, as aninhibitor of LCAT or can decrease LCAT activity by diluting theconcentration of the substrate phospholipid. Because SM is nothydrolyzed, it remains longer in the circulation. This feature permitscomplexes comprising Apo-SM to have longer duration of pharmacologicaleffect (mobilization of cholesterol) and to pick up more lipids, inparticular cholesterol. This will results in less frequent or smallerdoses necessary for treatment with Apo-SM particles.

[0063] The apolipoprotein(s) may be complexed with SM derived fromvirtually any source. For example, the SM may be obtained from milk, eggor brain. SM analogues or derivatives may also be used. Non-limitingexamples of useful SM analogues and derivatives include, but are notlimited to, palmitoylsphingomyelin and stearoylsphingomyelin.

[0064] The sphingomyelin may be artificially enriched in one particularsaturated or unsaturated acyl chain. For example, milk sphingomyelin(Avanti Phospholipid, Alabaster, Ala.) is characterized by longsaturated acyl chains. Milk sphingomyelin comprises about 20% of C16:0(16 carbon, saturated) acyl chain compared to the 80% comprised in eggsphingomyelin. Using solvent extraction, milk sphingomyelin can beenriched in one particular acyl chain to have a composition in acylchain comparable to, e.g., egg sphingomyelin. Acyl chains that may beutilized by the invention include, but are not limited to saturated acylchains (such as dipalmitoyl, distearoyl, diarachidonyl, and dibenzoylacyl chains), unsaturated chains (such as diolcoyl chains), mixed chainsof saturated and unsaturated acyl chains (such as palmitoyl or oleoylchains), saturated and/or unsaturated chains of mixed lengths, and etheranalogueues of saturated and unsaturated acyl chains.

[0065] The SM may be semi-synthetic such that it has a particular acylchain. For example, milk sphingomyelin can be first purified from milk,then one particular acyl chain, e.g., the C16:0 acyl chain, can becleaved and replaced by another acyl chain (preferably palmitic acid oroleic acid).

[0066] SM can also be entirely synthesized, by e.g., large-scalesynthesis. See, e.g., Dong et al, U.S. Pat. No. 5,220,043, entitledSynthesis of D-erythro-sphingomyelins, issued Jun. 15, 1993 ; Weis,1999, Chem. Phys. Lipids 102(1-2):3-12. Preferably, a predefinedsaturation level and fatty acid composition is selected for thesynthetic SM.

[0067] The complexes may optionally also include one or more otherphospholipids in addition to the SM. Virtually any type of phospholipidmay be used, including, but not limited to, small alkyl chainphospholipids, phosphatidylcholine (PC), egg phosphatidylcholine,soybean phosphatidylcholine, dipalmitoylphosphatidylcholine,dimyristoylphosphatidylcholine, distearoylphosphatidylcholine1-myristoy1-2-palmitoylphosphatidylcholine,1-palmitoy1-2-myristoylphosphatidylcholine,1-palmitoy1-2-stearoylphosphatidylcholine,1-stearoy1-2-palmitoylphosphatidylcholine,1-palmitoy1-2-oleoylphosphatidylcholine,1-oleoy1-2-palmitylphosphatidylcholine, dioleoylphosphatidylcholinedioleoylphosphatidylethanolamine, dilauroylphosphatidylglycerolphosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,phosphatidylinositol, sphingomyelin, sphingolipids,phosphatidylglycerol, diphosphatidylglycerol,dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol,distearoylphosphatidylglycerol, dioleoylphosphatidylglycerol,dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid,dimyristoylphosphatidylethanolamine,dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylserine,dipalmitoylphosphatidylserine, brain phosphatidylserine, brainsphingomyelin, dipalmitoylsphingomyelin, distearoylsphingomyelin,phosphatidic acid, galactocerebroside, gangliosides, cerebrosides,dilaurylphosphatidylcholine, (1,3)-D-mannosyl-(1,3)diglyceride,aminophenylglycoside, 3-cholesteryl-6′-(glycosylthio)hexyl etherglycolipids, and cholesterol and its derivatives. The compositions willtypically comprise about 40-85 wt% total phospholipid and about 60-15 wt% total apolipoprotein. (This range corresponds to roughly 1:25 to 1:200molar ratio of apolipoprotein to phospholipid). If an optional secondphospholipid is included, the sphingomyelin should typically comprisefrom about 25 to 75 wt % of the total phospholipid component, with thebalance being the second type of phospholipid.

[0068] The apolipoprotein-phospholipid complexes can be complexed inphospholipid:apolipoprotein ratios (Ri) that vary depending on theapolipoprotein and the nature of the SM and other phospholipid(s)comprised in the complex, as well as the expected size of the complexranging in size from 2-12 nm. For ApoA-I, the phospholipid:ApoA-I molarratio may vary from 25 to 200. The percentage of SM in the complex mayvary from 25% to 100% of the total phospholipid composition. Forexample, SM:PC:ApoA-I could be 25:25:1 (Ri=50) or 75:75:1 (Ri=150).Ratios in weight can be obtained by using a MW of 650-800 for thephospholipid.

[0069] The complexes may optionally also include paraoxonase (PON),antioxidants, cyclodextrins or other materials that help trapcholesterol in the core or the surface of the HDL-like particle. HDLparticle may optionally be pegylated (e.g., covered with polyethyleneglycol or other polymer) to increase circulation half-life.

[0070] Apolipoprotein, phospholipid, and Apo-phospholipid complexesutilized by the invention also include molecules that are labeled withany art-known detectable marker, including stable isotopes (e.g., ¹³C,¹⁵N, ²H, etc.); radioactive isotopes (e.g., ¹⁴C, ³H, ¹²⁵I, etc.);fluorophores; chemiluminescers; or enzymatic markers.

5.2. METHODS OF MAKING APO-SM COMPLEXES

[0071] Apo-SM complexes can be prepared in a variety of forms,including, but not limited to vesicles, liposomes, proteoliposomes,micelles, and discoidal particles. A variety of methods well known tothose skilled in the art can be used to prepare the Apo-SM complexes. Anumber of available techniques for preparing liposomes orproteoliposomes may be used. For example, apolipoprotein can beco-sonicated (using a bath or probe sonicator) with the appropriatelipid (i.e.,sphingomyelin) to form complexes. Alternatively,apolipoprotein can be combined with preformed lipid vesicles resultingin the spontaneous formation of Apo-SM complexes. The Apo-SM complexescan also be formed by a detergent dialysis method; e.g., a mixture ofApo, SM and a detergent such as cholate is dialyzed to remove thedetergent and reconstituted to form Apo-SM complexes (see, e.g., Jonaset al., 1986, Methods Enzymol. 128:553-82), or by using an extruderdevice or by homogenization.

[0072] In one embodiment, Apo-SM complexes can be prepared by thecholate dispersion method as described in Section 6.1 (Example 1).Briefly, dry lipid is hydrated in NaHCO₃ buffer, then vortexed andsonicated until all lipid is dispersed. Cholate solution is added, themixture is incubated for 30 minutes, with periodic vortexing andsonicating, until it turns clear, indicating that the lipid cholatemicelles are formed. ProApoA-I in NaHCO₃ buffer is added, and thesolution incubated for 1 hour at approximately 37° C.-50° C. The ratioof lipid:proApoA-I in the solution can be from 1:1 to 200:1 (mole/mole),but in a preferred embodiment, the ratio is 2:1 weight of lipid toweight of protein (wt/wt).

[0073] Cholate can be removed by methods well known in the art. Forexample cholate can be removed by dialysis, ultrafiltration or byremoval of cholate molecules by adsorption absorption onto an affinitybead or resin. In one embodiment, the affinity beads, e.g., BIO-BEADS®(Bio-Rad Laboratories) are added to the preparation of Apo-lipidcomplexes and cholate to adsorb the cholate. In another embodiment, thepreparation, e.g., a micellar preparation of the Apo-SM complexes andcholate, is passed over a column packed with affinity beads.

[0074] In a specific embodiment, cholate is removed from a preparationof proApoA-I-lipid complexes by loading the preparation onto BIO-BEADS®within a syringe. The syringe is then sealed with barrier film andincubated with rocking at 4° C. overnight. Before use, the cholate isremove by injecting the solution through BIO-BEADS®, where it isadsorbed by the beads.

[0075] The Apo-SM complexes have an increased half-life in thecirculation when the complexes have a similar size and density to HDL,especially to the HDLs in the pre-β-1 or pre-β-2 HDL populations. Stablepreparations having a long shelf life may be made by lyophilization—theco-lyophilization procedure described below being a preferred approachdue to the stability of the resulting formulation and the ease offormulation/particle preparation process. Co-lyophilization methods arealso described in U.S. Pat. No. 6,287,590 (entitled Peptide/lipidcomplex formation by co-lyophilization, by Dasseux, issued Sep. 11,2001), which is incorporated herein by reference in its entirety. Thelyophilized Apo-SM complexes can be used to prepare bulk forpharmaceutical reformulation, or to prepare individual aliquots ordosage units that can be reconstituted by rehydration with sterile wateror an appropriate buffered solution prior to administration to asubject.

[0076] As disclosed above, apolipoprotein may be complexed with avariety of sphingomyelins. The sphingomyelin may be artificiallyenriched in one particular saturated or unsaturated acyl chain. Forexample, milk sphingomyelin (Avanti Phospholipid, Alabaster, Ala.) ischaracterized by long saturated acyl chains. Milk sphingomyelincomprises about 20% of C16:0 (16 carbon, saturated) acyl chain comparedto the 80% comprised in egg sphingomyelin. Using solvent extraction,milk sphingomyelin can be enriched in one particular acyl chain to havea composition in acyl chain comparable to, e.g., egg sphingoniyelin.Acyl chains that may be utilized by the invention include, but are notlimited to, saturated acyl chains (such as dipalmitoyl, distearoyl,diarachidonyl, and dibehenoyl acyl chains), unsaturated chains (such asdioleoyl chains), mixed chains of saturated and unsaturated acyl chains(such as palmitoyl or oleyl chains), saturated and/or unsaturated chainsof mixed lengths, and ether analogueues of saturated and unsaturatedacyl chains.

[0077] In certain preferred embodiments, a source of naturalsphingomyelin is selected to avoid contamination by prions (e.g., inbrain sphingomyelin) or viral contamination (e.g., in eggsphingomyelin). In another preferred embodiment, fully synthetic SM isselected to avoid contamination. In other embodiments, a SM with a highsaturation state is used to improve chemical stability of the Apo-AI-SMcomplex.

[0078] The SM may be semi-synthetic such that it has a particular acylchain. For example, milk sphingomyelin can be first purified from milk,then one particular acyl chain, e.g., the C16:0 acyl chain, can becleaved and replaced by another acyl chain (preferably palmitic acid oroleic acid).

[0079] SM can also be entirely synthesized, by e.g., large-scalesynthesis (see, e.g., Dong et al., U.S. Pat. No. 5,220,043, entitledSynthesis of D-erythro-sphingomyelins, issued Jun. 15, 1993 ; Weis,1999, Chem. Phys. Lipids 102(1-2):3-12). Preferably, a predefinedsaturation level and fatty acid composition is selected for thesynthetic SM.

[0080] U.S. Pat. Nos. 6,004,925, 6,037,323, 6,046,166 and 6,287,590(incorporated herein by reference in their entireties) disclose a simplemethod for preparing apolipoprotein-lipid (Apo-lipid) complexes thathave characteristics similar to HDL. This preferred method,co-lyophilization of Apo and lipid solutions in organic solvent (orsolvent mixtures) and formation of Apo-lipid complexes during hydrationof the lyophilized powder, has the following advantages: (1) The methodrequires very few steps. (2) The method uses inexpensive solvent(s). (3)Most or all of the included ingredients are used to form the designedcomplexes, thus avoiding waste of starting material that is common tothe other methods. (4) Lyophilized compounds are formed that are verystable during storage. The resulting complexes may be reconstitutedimmediately before use. (5) The resulting complexes usually need not befurther purified after formation and before use. (6) Toxic compounds,including detergents such as cholate, are avoided. (7) The productionmethod can be easily scaled up and is suitable for GMP manufacture(i.e., in an endotoxin-free environment).

[0081] In a preferred embodiment, co-lyophilization methods commonlyknown in the art are used to prepare Apo-SM complexes. Briefly, theco-lyophilization steps include solubilizing Apo and lipid in organicsolvent of solvent mixture, or solubilizing Apo and lipid separately andmixing them together. The desirable characteristics of solvent orsolvent mixture are: (i) a medium relative polarity to be able todissolve hydrophobic lipids and amphipatic protein, (ii) solvents shouldbe class 2 or 3 solvent according to FDA solvent guidelines (FederalRegister, volume 62, No. 247) to avoid potential toxicity associatedwith the residual organic solvent, (iii) low boiling point to assureease of solvent removal during lyophilization, (iv) high melting pointto provide for faster freezing, higher temperatures of condenser and,hence less ware of freeze-dryer. In a preferred embodiment, glacialacetic acid is used. Combinations of e.g., methanol, glacial aceticacid, xylene, or cyclohexane may also be used.

[0082] The Apo/Lipid solution is then lyophilized to obtain homogeneousApo/lipid powder. The lyophilization conditions can be optimized toobtain fast evaporation of solvent with minimal amount of residualsolvent in the lyophilized Apo/lipid powder. The selection offreeze-drying conditions can be determined by the skilled artisan, anddepends on the nature or solvent, type and dimensions of the receptacle,e.g., vial, holding solution, fill volume, and characteristics offreeze-dryer used. The concentration of lipid/Apo solution prior to thelyophilization, for organic solvent removal and successful formation ofcomplexes, is preferably 10 to 50 mg/ml concentration of Apo and 20 to100 mg/ml concentrations of lipid.

[0083] The Apo-lipid complexes form spontaneously after hydration ofApo-lipid lyophilized powder with an aqueous media of appropriate pH andosmolality. In some embodiments, the media may also contain stabilizerssuch as sucrose, trehalose, glycerin and others. In some embodiments,the solution must be heated several times above transition temperaturefor lipids for complexes to form. The ratio of lipid to protein forsuccessful formation of Apo-SM complexes can be from 1:1 to 200:1(mole/mole), and is preferably 2:1 weight of lipid to weight of protein(wt/wt). Powder is hydrated to obtain final complex concentration of5-30 mg/ml expressed in protein equivalents.

[0084] In one embodiment, Apo powder is obtained by freeze-drying Aposolution in NH₄HCO₃ aqueous solution. A homogeneous solution of Apo andlipid (i.e., sphingomyelin) is formed by dissolving their powders andApo in glacial acetic acid. The solution is then lyophilized, andHDL-like Apo-lipid complexes are formed by hydration of lyophilizedpowder with aqueous media.

[0085] The another preferred method is homogenization. This method maybe used to prepare Apo soybean-PC complexes and is routinely used forformulation of AI-_(Milano)-POPC complexes. Homogenization can be easilyadapted for formation of Apo-SM complexes. Briefly, this methodcomprises forming a suspension of lipids in aqueous solution of Apo byUltraturex™, and homogenization of formed lipid-protein suspension usinghigh-pressure homogenizer until suspension becomes clear-opalescentsolution and complexes are formed. Elevated temperatures above lipidtransition are used during homogenization. Solution is homogenized forextended period of time 1-14 hours and elevated pressure.

[0086] In another embodiment, Apo-SM complexes are formed byco-lyophilization of phospholipid with peptide or protein solutions orsuspensions. The homogeneous solution of peptide/protein and SM (plusany other phospholipid of choice) in an organic solvent or organicsolvent mixture can be lyophilized, and Apo-SM complexes can be formedspontaneously by hydration of the lyophilized powder with an aqueousbuffer. Examples of organic solvents or their mixtures are include, butare not limited to, acetic acid, acetic acid and xylene, acetic acid andcyclohexane, and methanol and xylene.

[0087] A suitable proportion of protein (peptide) to lipid can bedetermined empirically so that the resulting complexes possess theappropriate physical and chemical properties; i.e., usually (but notnecessarily) similar in size to HDL. The resulting mixture of Apo andlipid in solvent is frozen and lyophilized to dryness. Sometimes anadditional solvent must be added to the mixture to facilitatelyophilization. This lyophilized product can be stored for long periodsand will remain stable.

[0088] The lyophilized product can be reconstituted in order to obtain asolution or suspension of the Apo-lipid complex. To this end, thelyophilized powder is rehydrated with an aqueous solution to a suitablevolume (typically 5-20 mg Apo-SM complex/ml) which is convenient fore.g., intravenous injection. In a preferred embodiment the lyophilizedpowder is rehydrated with phosphate buffered saline, saline bicarbonate,or a physiological saline solution. The mixture may be agitated orvortexed to facilitate rehydration. In general, the reconstitution stepshould be conducted at a temperature equal to or greater than the phasetransition temperature of the lipid component of the complexes. Withinminutes of reconstitution, a clear preparation of reconstitutedApo-lipid complexes will result.

[0089] An aliquot of the resulting reconstituted preparation can becharacterized to confirm that the complexes in the preparation have thedesired size distribution; e.g., the size distribution of HDL.Characterization of the reconstituted preparation can be performed usingany method known in the art, including, but not limited to, sizeexclusion filtration, gel filtration, column filtration, and gelpermeation chromatography.

[0090] For example, after hydration of lyophilized Apo-lipid powder orat the end of homogenization or cholate dialysis formed Apo-lipidHDL-like particles are characterized with respect to their size,concentration, final pH and osmolality of resulting solution, in someinstances integrities of lipid and apolipoprotein are characterized. Thesize of the resulting Apo-lipid particles is determinative of theirefficacy, therefore making this measurement is preferred forcharacterization of the particles.

[0091] In one embodiment, gel permeation chromatography (GPC), e.g., ahigh pressure liquid chromatography system equipped with a 1×30 cmSuperdex™ column (Pharmacia Biotech) and UV-detector may be used.Complexes are eluted with bicarbonate buffered saline comprised of 140mM NaCl and 20 mM sodium bicarbonate delivered with 0.5 ml/min flowrate. A typical amount of complex injected is 0.1 to 1 mg based onprotein weight. The complexes are monitored by absorbance at 280 nm.

[0092] Protein and lipid concentration of Apo-lipid particles solutioncan be measured by any method known in the art, including, but notlimited to, protein and phospholipid assays as well as bychromatographic methods such as HPLC, gel filtration chromatography, GCcoupled with various detectors including mass spectrometry, UV ordiode-array, fluorescent, elastic light scattering and others. Theintegrity of lipid and proteins can be also determined by the samechromatographic techniques as well as peptide mapping, SDS-page gel, N-and C-terminal sequencing for proteins and standard assays to determinelipid oxidation for lipids.

5.3 PHARMACEUTICAL COMPOSITIONS

[0093] The pharmaceutical compositions utilized by the inventioncomprise Apo-SM complexes as the active ingredient in a pharmaceuticallyacceptable carrier suitable for administration and delivery in vivo.Since peptides may comprise acidic and/or basic termini and/or sidechains, peptides can be included in the compositions in either the formof free acids or bases, or in the form of pharmaceutically acceptablesalts.

[0094] Injectable compositions include sterile suspensions, solutions oremulsions of the active ingredient in aqueous or oily vehicles. Thecompositions can also comprise formulating agents, such as suspending,stabilizing and/or dispersing agent. The compositions for injection canbe presented in unit dosage form, e.g., in ampules or in multidosecontainers, and can comprise added preservatives. For infusion, acompositions is preferably supplied in an infusion bag made of materialcompatible with Apo-lipid complexes, such as ethylene vinyl acetate orany other compatible material known in the art.

[0095] Alternatively, the injectable compositions can be provided inpowder form for reconstitution with a suitable vehicle, including butnot limited to, sterile pyrogen free water, buffer, dextrose solution,etc., before use. To this end, Apo can be lyophilized, or co-lyophilizedApo-SM complexes may be prepared. The stored compositions can besupplied in unit dosage forms and reconstituted prior to use in vivo.

[0096] For prolonged delivery, the active ingredient can be formulatedas a depot composition, for administration by implantation; e.g.,subcutaneous, intradermal, or intramuscular injection. Thus, forexample, Apo-lipid complex or Apolipoprotein alone may be formulatedwith suitable polymeric or hydrophobic materials (e.g., as an emulsionin an acceptable oil) or in phospholipid foam or ion exchange resins.

[0097] Alternatively, transdermal delivery systems manufactured as anadhesive disc or patch that slowly releases the active ingredient forpercutaneous absorption can be used. To this end, permeation enhancerscan be used to facilitate transdermal penetration of the activeingredient. A particular benefit can be achieved by incorporating theApo-SM complexes utilized by the invention into a nitroglycerin patchfor use in patients with ischemic heart disease andhypercholesterolemia.

[0098] The compositions can, if desired, be presented in a pack ordispenser device that may comprise one or more unit dosage formscomprising the active ingredient. The pack can for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice can be accompanied by instructions for administration.

5.4 METHODS OF TREATMENT

[0099] The Apo-SM complexes utilized by the invention can be used totreat or prevent virtually any disease, condition or disorder responsiveto apolipoproteins or other apolipoprotein-phospholipid particles (e.g.,ApoAI-SoybeanPC, ApoAI-POPC), including but not limited to, coronaryheart disease; coronary artery disease; cardiovascular disease,hypertension, restenosis, vascular or perivascular diseases;dyslipidemic disorders; dyslipoproteinemia; high levels of low densitylipoprotein cholesterol; high levels of very low density lipoproteincholesterol; low levels of high density lipoproteins; high levels oflipoprotein Lp(a) cholesterol; high levels of apolipoprotein B;atherosclerosis (including treatment and prevention of atherosclerosis);hyperlipidemia; hypercholesterolemia; familial hypercholesterolemia(FH); familial combined hyperlipidemia (FCH); lipoprotein lipasedeficiencies, such as hypertriglyceridemia, hypoalphalipoproteinemia,and hypercholesterolemialipoprotein.

[0100] Using the methods of the invention, a dosage of apolipoproteinfrom 2- to 25-fold less than the effective dosage currently known in theart would be expected to be efficacious in treating or preventing thedisease or in bringing about an ameliorative effect.

[0101] In one embodiment, the methods of the invention encompass amethod of treating or preventing a disease associated with dyslipidemia,comprising administering to a subject a composition comprising anapolipoprotein and sphingomyelin in an amount effective to achieve aserum level of free or complexed apolipoprotein in the range of 10 mg/dLto 300 mg/dL above a baseline (initial) level before administrationbetween 5 minutes and 1 day after administration.

[0102] In another embodiment, the methods of the invention encompass amethod of treating or preventing a disease associated with dyslipidemia,comprising administering to a subject an apolipoprotein-sphingomyelincomplex in an amount effective to achieve a circulating plasmaconcentrations of a HDL-cholesterol fraction between 10% and 1000% ofthe initial HDL-cholesterol fraction concentration between 5 minutes and1 day after administration.

[0103] In another embodiment, the methods of the invention encompass amethod of treating or preventing a disease associated with dyslipidemia,comprising administering to a subject an apolipoprotein-sphingomyelincomplex in an amount effective to achieve a circulating plasmaconcentration of a HDL-cholesterol fraction between 30 and 300 mg/dLbetween 5 minutes and 1 day after administration.

[0104] In another embodiment, the methods of the invention encompass amethod of treating or preventing a disease associated with dyslipidemia,comprising administering to a subject an apolipoprotein-sphingomyelincomplex in an amount effective to achieve a circulating plasmaconcentrations of cholesteryl esters between 30 and 300 mg/dL between 5minutes and 1 day after administration.

[0105] The Apo-SM complexes can be used alone or in combination therapywith other drugs used to treat or prevent the foregoing conditions.Such-therapies include, but are not limited to simultaneous orsequential administration of the drugs involved. For example, in thetreatment of hypercholesterolemia or atherosclerosis, the Apo-SMformulations can be administered with any one or more of the cholesterollowering therapies currently in use; e.g., bile-acid resins, niacin,statins and/or fibrates. Such a combined regimen may produceparticularly beneficial therapeutic effects since each drug acts on adifferent target in cholesterol synthesis and transport; i.e., bile-acidresins affect cholesterol recycling, the chylomicron and LDL population;niacin primarily affects the VLDL and LDL population; the statinsinhibit cholesterol synthesis, decreasing the LDL population (andperhaps increasing LDL receptor expression); whereas the Apo-SMcomplexes affect RCT, increase HDL, and promote cholesterol efflux.

[0106] In another embodiment, the Apo-SM complexes may be used inconjunction with fibrates to treat or prevent coronary heart disease;coronary artery disease; cardiovascular disease, hypertension,restenosis, vascular or perivascular diseases; dyslipidemic disorders;dyslipoproteinemia; high levels of low density lipoprotein cholesterol;high levels of very low density lipoprotein cholesterol; low levels ofhigh density lipoproteins; high levels of lipoprotein Lp(a) cholesterol;high levels of apolipoprotein B; atherosclerosis (including treatmentand prevention of atherosclerosis); hyperlipidemia;hypercholesterolemia; familial hypercholesterolemia (FH); familialcombined hyperlipidemia (FCH); lipoprotein lipase deficiencies, such ashypertriglyceridemia, hypoalphalipoproteinemia, andhypercholesterolemialipoprotein. Exemplary formulations and treatmentregimens are described below.

[0107] The Apo-SM complexes utilized by the invention may beadministered by any suitable route that ensures bioavailability in thecirculation. An important feature of the invention is that Apo-SMcomplexes may be administered in doses less than 1-10% of the effectivedose required for apolipoprotein (Apo) or Apo peptide administeredalone, and in doses 2-25 fold less than the effective dose required forApo-soybean PC (or Apo-egg PC or Apo-POPC) administration.Administration at doses (for intravenous injection) as low as about 40mg to 2 g/person of apolipoprotein every 2 to 10 days is required,rather than the large amounts of apolipoprotein (20 mg/kg to 100 mg/kgper administration every 2 to 5 days, 1.4 g to 8 g per average sizedhuman) required by currently available treatment regimens.

[0108] The Apo-SM complexes utilized by the invention are administeredin dosages that increase the small HDL fraction, and preferably, the preβ- (and pre-γ), and pre-β-like HDL fraction.

[0109] Administration can best be achieved by parenteral routes ofadministration, including intravenous (IV), intramuscular (IM),intradermal, subcutaneous (SC), and intraperitoneal (IP) injections. Incertain embodiments, administration is by a perfuser, an infiltrator ora catheter. In a preferred embodiments, the Apo-SM complexes areadministered by injection, by a subcutaneously implantable pump or by adepot preparation, in amounts that achieve a circulating serumconcentration equal to that obtained through parenteral administration.

[0110] Administration can be achieved through a variety of differenttreatment regimens. For example, several intravenous injections can beadministered periodically during a single day, with the cumulative totalvolume of the injections not reaching the daily toxic dose.Alternatively, one intravenous injection can be administered about every3 to 15 days, preferably about every 5 to 10 days, and most preferablyabout every 10 days. In yet another alternative, an escalating dose canbe administered, starting with about 1 to 5 doses at a dose between(50-200 mg) per administration, then followed by repeated doses ofbetween 200 mg and 1 g per administration. Depending on the needs of thepatient, administration can be by slow infusion with a duration of morethan one hour, by rapid infusion of one hour or less, or by a singlebolus injection.

[0111] Complexing apolipoprotein with SM rather than with another lipid,e.g., phosphatidyl choline (PC), increases its toxicity at a givenindividual dose, because the complex removes more cholesterol from serumcirculation, allowing it to have greater pharmacological efficacy andtoxic effects. For example, if apolipoprotein-SM is parenterallyadministered, e.g., by injection, then it is toxic at a dose of 200mg/kg. However, if injections are given at a lower dosage for severaldays, then gradually increased, the recipient will adapt to theadministration and a higher final dosage can be achieved. For example,an injection can be administered on two sequential days at aconcentration of 10 mg/kg, followed by injection on the next twosequential days at a concentration of 20 mg/kg, followed by injection onthe next two sequential days at a concentration of 30 mg/kg followed byinjection on subsequent days of 40 mg/kg per day. Under such a schedule,apolipoprotein-SM complexes can be administered for up to two weekswithout problems of toxicity.

[0112] Other routes of administration may be used. For example,absorption through the gastrointestinal tract can be accomplished byoral routes of administration (including but not limited to ingestion,buccal and sublingual routes) provided appropriate formulations (e.g.,enteric coatings) are used to avoid or minimize degradation of theactive ingredient, e.g., in the harsh environments of the oral mucosa,stomach and/or small intestine. Alternatively, administration viamucosal tissue such as vaginal and rectal modes of administration may beutilized to avoid or minimize degradation in the gastrointestinal tract.In yet another alternative, the formulations of the invention can beadministered transcutaneously (e.g., transdermally), or by inhalation.It will be appreciated that the preferred route may vary with thecondition, age and compliance of the recipient.

[0113] The actual dose of Apo-SM complexes will vary with the route ofadministration. In one embodiment, the dose is adjusted to achieve aserum level of free or complexed apolipoprotein in the range of 10 mg/dlto 300 mg/dl above a baseline (initial) level before administrationbetween 5 minutes and 1 day after administration. The baseline level isthe initial apolipoprotein level prior to administration of the Apo-SMcomplexes.

[0114] In another embodiment, the dose is adjusted to achieve acirculating plasma concentrations of a HDL-cholesterol fraction between10% and 1000% of the initial HDL-cholesterol fraction concentrationbetween 5 minutes and 1 day after intravenous administration.

[0115] In another embodiment, the dose is adjusted to achieve atemporary or permanent circulating plasma concentrations of aHDL-cholesterol fraction between 30 and 300 mg/dl between 5 minutes and1 day after intravenous administration.

[0116] In another embodiment, the dose is adjusted to achieve atemporary or permanent circulating plasma concentrations of cholesterylesters between 30 and 300 mg/dl between 5 minutes and 1 day afterintravenous administration.

[0117] Data obtained in animal model systems described in U.S. Pat. Nos.6,004,925, 6,037,323 and 6,046,166 (issued to Dasseux et al.,incorporated herein by reference in their entireties) show that ApoA-Ipeptides associate with the HDL component, and have a projectedhalf-life in humans of about five days. Thus, in one embodiment, Apo-SMcomplexes can be administered by intravenous injection at a dose betweenabout 0.1 g-1 g of Apo-SM per administration every 2 to 10 days peraverage sized human.

[0118] Toxicity and therapeutic efficacy of the various Apo-SM complexescan be determined using standard pharmaceutical procedures in cellculture or experimental animals for determining the LD₅₀ (the doselethal to 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio LD₅₀ /ED₅₀. Apo-SM complexes that exhibit large therapeuticindices are preferred.

[0119] Patients can be treated from a few days to several weeks before amedical act (e.g., preventive treatment), or during or after a medicalact. Administration can be concomitant to or contemporaneous withanother invasive therapy, such as, angioplasty, carotid ablation,rotoblader or organ transplant (e.g., heart, kidney, liver, etc.).

[0120] In certain embodiments, Apo-SM complexes are administered to apatient whose cholesterol synthesis is controlled by a statin or acholesterol synthesis inhibitor. In other embodiments, Apo-SM complexesare administered to a patient undergoing treatment with a binding resin,e.g., a semi-synthetic resin such as cholestyramine, or with a fiber,e.g., plant fiber, to trap bile salts and cholesterol, to increase bileacid excretion and lower blood cholesterol concentrations.

5.5 OTHER USES

[0121] The Apo-SM complexes utilized by the invention can be used inassays in vitro to measure serum HDL, e.g., for diagnostic purposes.Because ApoA-I, ApoA-II and Apo peptides associate with the HDLcomponent of serum, Apo-SM complexes can be used as “markers” for theHDL population, and the pre-β1 and pre-β2 HDL populations. Moreover, theApo-SM complexes can be used as markers for the subpopulation of HDLthat are effective in RCT. To this end, the Apo-SM can be added to ormixed with a patient serum sample; after an appropriate incubation time,the HDL component can be assayed by detecting the incorporated Apo-SM.This can be accomplished using labeled Apo-SM (e.g., radiolabels,fluorescent labels, enzyme labels, dyes, etc.), or by immunoassays usingantibodies (or antibody fragments) specific for Apo-SM.

[0122] Alternatively, labeled Apo-SM can be used in imaging procedures(e.g., CAT scans, MRI scans) to visualize the circulatory system, or tomonitor RCT, or to visualize accumulation of HDL at fatty streaks,atherosclerotic lesions, and the like, where the HDL should be active incholesterol efflux.

6. EXAMPLES 6.1. EXAMPLE 1

[0123] Preparation of ProApoA-I-Lipid Complexes

[0124] Complexes of proApoA-I and phospholipids are drug candidates thatpotentially mimic the biological activities of HDL. This exampledescribes the preparation of proApoA-I-phospholipid complexes.

6.1.1. MATERIALS AND METHODS

[0125] Complexes of proApoA-I and lipids were prepared by the cholatedispersion method. Four lipids were used: Phospholipon 90 G (soybeanPC), sphingomyelin, dipalmitoyl phosphatidylcholine (DPPC), and1-palmitoyl-2-oleyl-phosphatidylcholine (POPC).

[0126] A 30 mg/ml cholate solution was made by adding 0.9677 g of cholicacid to 32.25 ml of 2 mM NaHCO₃ buffer.

[0127] To prepare the proApoA-I solution, proApoA-I solution(Eurogentec) at a concentration of 1 mg/ml in 6M urea was employed. 60ml of the proApoA-I-6M urea solution was dialyzed against 1200 ml of 2mM NaHCO₃ buffer using a tangential flow filtration unit (Labscale TFFSystem, Millipore) equipped with a 10 kDa cut off membrane. Followingdialysis, the solution was concentrated to a final volume of 20 ml.Remaining solution was collected and the protein concentration wasdetermined to be 2.62 mg/ml solution by a Markwell-Lowry protein assay(Markwell et al., 1978, Anal. Biochem. 87(1): 206-10).

[0128] To prepare the Phospholipon 90 G (soybean PC) solution,Phospholipon 90 G (Rhône-Poulenc Nattermann Phospholipid GMBH # 228154)was aliquoted into 50 ml aliquots and stored under N₂ gas at −20° C. A25 mg/ml Phospholipon 90 G solution was made by adding 0.7815 g ofPhospholipon 90 G to 31.26 ml of chloroform.

[0129] A 25 mg/ml sphingomyelin solution was made by adding 0.0637 g ofsphingomyelin to 2.548 ml of chloroform.

[0130] 25 mg/ml solutions of 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine(DPPC) and 1-palmitoyl-2-oleyl-phosphatidylcholine (POPC) in chloroformwere also prepared.

[0131] 250 μl aliquots of the Phospholipon 90 G, sphingomyelin, DPPC andPOPC solutions were then placed in glass tubes and the chloroformevaporated by N₂ blowing and vortexing to produce 6.25 mg of dry lipidper tube. 400 μl of 2 mM NaHCO₃ buffer was added to each tube and thelipids were hydrated for 15 min at 37° C., for all the lipids exceptDPPC, which was hydrated at 50° C. for 15 min. The tubes of solutionwere vortexed and sonicated periodically until all lipids weredispersed. 160 μl of the 30 mg/ml cholate solution was added to eachtube.

[0132] 160 ml of the 30 mg/ml cholate solution was added to each tube toclarify the sphingomyelin solution. The lipid-cholate solutions wereincubated at 37° C. (for POPC, SM, and soybean PC) and 50° C. (forDPPC). During incubation, solutions were periodically vortexed andsonicated. If solutions would not clear after vortexing and sonicatingsteps, then additional cholate solution was added to the lipid-cholatesolution in a series of 100 ml aliquots. Incubation, vortex, sonicationand addition of cholate continued until all solutions became clear.Total cholate solution added was 160, 260, 560 and 560 ml for the SM,DPPC, soybean PC, and POPC, respectively. The lipid-cholate solutionswere incubated for a total of 30 min.

[0133] 1.908 ml of the 2.62 mg/ml solution of proApoA-I in 2 mM NaHCO₃buffer was added to each tube. The ratio of proApoA-I:lipid in thesolution was 5 mg:6.25 mg or 1:1.25 (wt/wt). The proApoA-1-lipid-cholatesolutions were incubated for 1 h at 37° C. (for POPC, SM, and soybeanPC) and at 50° C. (for DPPC).

[0134] To remove cholate, which is toxic, the solutions were incubatedwith ion exchange resin (BIO-BEADS®, Sigma). BIO-BEADS® were activatedby incubation of one volume of dry BIO-BEADS® with two volumes of 2 mMNaHCO₃ for 6 hours. Ten ml and 30 ml plastic syringes were loaded withactivated BIO-BEADS®. The preparation of proApoA-I-lipid complexes wasthen loaded onto the BIO-BEADS®. For the sphingomyelin and DPPCsolutions, approximately 5-7 ml of BIO-BEADS® were added per 4.8 mg ofcholate solution. For soybean PC and POPC, approximately 12 ml ofBIO-BEADS® were added per 16.8 mg of cholate solution. TheproApoA-I-lipid comnplex solutions were then loaded on the BIO-BEADS®within the syringes. The syringes were then sealed with barrier film andincubated with rocking at 4° C. overnight. At the end of incubation,solutions of the proApo-A-I-lipid complexes were collected and filteredthrough 0.22 mm PES filters. Solutions were then stored at 4° C. priorto the analysis.

[0135] Complex solutions were analyzed using GPC (gel permeationchromatography) using a 1×30 cm Superdex™ 200 (Pharmacia Biotech)column. An aqueous mobile phase containing 20 mM NaHCO₃ (pH 8) wasdelivered at a 0.5 ml/min flow rate. The injection volume was 100 μl.The run time was 45 min. Complex was detected by absorption at 280 nm.The retention time of the proApoA-I-lipid complex was compared with theretention time of purified rabbit HDL.

6.1.2. RESULTS AND DISCUSSION

[0136] Chromatograms of proApo-A-I complexes contained two major peaks:the first, appearing at around 22 min, corresponded to a molecularweight of roughly 200-300 kDa. The second, appearing at around 26 min,corresponded to a molecular weight of roughly 100 kDa. In eachchromatogram, the peak at 22 min corresponded to lipid-rich proApo-A-Ilipid complexes, while the peak at 26 min corresponded to lipid poorproApo-A-I lipid complexes. The chromatogram of purified rabbit HDLstandard produced a single peak at around 20 min. Calculated from theretention time of the purified rabbit HDL, the formulation was optimizedto yield a monomodal peak at around 22 min of lipid-rich proApo-AIcomplexes.

[0137] The ratio between the 22 min and 26 min peaks was differed fordifferent lipids. For proApo-A-I-POPC and proApo-A-I-soybean PCcomplexes at a ratio of 1:1.25 protein/lipid (wt/wt), the fraction of 26min peak area, in relation to the total area, was only 10-20%.Therefore, no further optimization was required for these complexes. Incontrast, for proApo-A-I-SM and proApo-A-I DPPC complexes at 1:1.25(wt/wt) protein/lipid ratio, the fraction of 26 min peak area inrelation to the total area was 30-50%. The difference between 22 min and26 min peak ratio for the different lipids could be attributed thevariations in molecular weight and structure of the lipids, and,therefore, different wt/wt ratios of protein to lipid required to formlipid-rich complexes. In order to increase the fraction of lipid-rich,22 min peak for proApo-A-I SM complexes, the wt/wt ratio of SM toprotein was increased in Example 2 below.

6.2. EXAMPLE 2

[0138] Further Preparation of ProApoA-I-Lipid Complexes

[0139] Based on the results obtained in Section 6.1 (Example 1), theproportion of SM was increased, in order to produce preferentially asingle peak of proApoA-I-SM complexes. This example demonstrates thatthe proApoA-I to sphingomyelin ratio may be varied to produce a singlepeak of proApoA-I-sphingomyelin complex at a ratio of 1:2 (wt/wt).

6.2.1. MATERIALS AND METHODS

[0140] ProApoA-I -SM complexes were prepared by the followingco-solubilization method.

[0141] ProApoA-I solution in 6M urea (Eurogentec) was concentrated 5times and dialyzed against 10 volumes of 5 mM NH₄HCO₃. The proteinconcentration was then measured by performing a Markwell-Lowry proteinassay. Protein solution was then lyophilized.

[0142] A 25 mg/ml stock solution of proApoA-I in acetic acid was made bydissolving 128.2 mg of the lyophilized proApo-A-I powder in 5.13 ml ofglacial acetic acid (J T Baker).

[0143] A 50 mg/ml stock solution of sphingomyelin was made by dissolving256.0 mg of sphingomyelin (Avanti) in 5.12 ml of glacial acetic acid.

[0144] Solutions of proApoA-I:SM were combined to weight:weight (wt/wt)ratios of 1: 1, 1:1.25, 1:1.5 and 1:2 by combining 1.0 ml of proApo-A-Istock solution with 0.5, 0.625, 0.75 and 1.0 ml of sphingomyelin-stocksolution. The combined solutions were filtered using a syringe-driven0.2 μm polyethersulfone filter (Whatman) and the volume of filteredsolution was recorded.

[0145] Solutions of proApoA-I-SM were frozen at −40° C. forapproximately 1.5 hours, then lyophilized in a freeze-dryer as indicatedat the temperatures and in the order shown below: Temperature Time −20°C.   2 h −20° C. to 23° C.  12 h   50° C.   6 h   23° C. 1.5 h

[0146] ProApoA-I SM complexes were formed by hydrating the lyophilizedpowder in bicarbonate saline (140 mM NaCl, 20 mM NaHCO₃) to aconcentration of 10 mg/ml protein. Solutions were heated to 52° C. andcooled to room temperature three times in order to facilitate hydration.

[0147] Aliquots of 200 μl of complex solutions were diluted using 200 μlbicarbonate saline and analyzed on a Superdex 200 HR 10/30 GPC column(Amersham Pharmacia Biotech AB) using the following conditions. Runningbuffer was 140 mM NaCl and 20 mM NaHCO₃. The flow rate was 0.5 ml/min.The run time was 50 min. The injection volume was 100 μl. Detectionwavelength was 220 nm. The retention times of complexes was compared tothe retention time of a gel filtration standard (Bio-Rad).

6.2.2. RESULTS AND DISCUSSION

[0148] Chromatograms contained two major peaks at 24 and 29 min. Thepeak at 24 min corresponded to proApoA-I-SM complexes while the peak at29 min corresponded to free proApoA-I protein. The ratio between the 24and 29 min peaks differed depending upon the ratio of proApo-A-I-SM witha greater percentage of the peak appearing in the complex portion as thewt/wt ratio increased. At 1:2 wt/wt of proApoA-I :SM, a single peak at24 min was observed, hence, homogeneous distribution of-complex size wasobtained.

6.3. EXAMPLE 3

[0149] Pharmacological Efficacy of r-proApoA-I-Sphingomyelin ComplexesCompared With r-proApoA-I-POPC Complexes

[0150] ProApoA-I-lipid complexes are drug candidates that potentiallymimic the biological activities of HDL. The objective of this study wasto compare the pharmacodynamic properties (i.e., mobilization ofcholesterol in plasma) of proApoA-I-SM with those of conventionalproApoA-I-phosphotidyl choline complexes such as proApoA-I-POPC.Recombinant human proApoA-I (r-proApoA-I) was used in this study.Proapolipoprotein A-I comprises six amino acids(Arg-His-Phe-Trp-Gln-Glu) attached at the amino terminal end of ApoA-I.

6.3.1. MATERIALS AND METHODS

[0151] Complex Formulation

[0152] ProApoA-I-SM and proApoA-I-POPC complexes were prepared by thecholate dialysis method as described above. The ratio of apolipoproteinto lipid was 1:2 (weight of protein/weight of lipid) for proApoA-I-SMand proApo-A-I-POPC. The protein r-proApoA-I (Eurogentec, Belgum)solution in 1 mg/ml in 6M urea was dialyzed with 20 volumes of 5 mMNH₄HCO₃ solution (pH 8). Protein concentration after dialysis wasdetermined by a Modified Lowry Assay.

[0153] Sodium cholate (Sigma) was added to the protein solution at a 1:2ratio (weight of r-proApoA-I/weight of sodium cholate). Thenphospholipids were added to the protein-cholate solutions at a 1:2weight ratio of apolipoprotein to phospholipid.Protein-cholate-phospholipid solutions were incubated at 37° C. forsolutions containing POPC and 50° C. for solutions containing SM. Duringincubation solutions were periodically mixed by vortexing.

[0154] After 1 hour of incubation, clear solutions were formed. Cholate,which is toxic for human and animals, was removed from solutions byincubation in presence of an ion exchange resin Biobeads™ (BioRad).Biobeads™ were added to protein-phospholipid-cholate solution at 1:2ratio of hydrated Biobeads™ volume to solution volume. The resultingsolutions were incubated for 12 hours at 4° C. under rocking. During theincubation, cholate was absorbed by Biobeads™ ion exchange resin.Removal of cholate resulted in formation of cholate freeapolipoprotein-phospholipid complexes. At the end of incubationsolutions were filtered to remove Biobeads™ and the resulting solutionswere sterilized. Trehalose was added to the final solution at 80 mg/mlto adjust osmolality. The resulting solutions were analyzed and storedat 4° C. prior to injection into the animals.

[0155] Complex Characterization

[0156] After formulation, apolipoprotein-phospholipid complexes wereanalyzed to determine their size, protein concentration, solution pH andosmolality. Complex sizes were analyzed using GPC (gel permeationchromatography) equipped with a 1×30 cm Superdex™ 200 (PharmaciaBiotech) column. An aqueous mobile phase containing 20 mM NaHCO₃ (pH 8)was delivered at a 0.5 ml/min flow rate. The injection volume was 100μl. Complexes were detected by UV absorption at 280 nm. Proteinconcentration was determined by routine methods (i.e., a modifiedMarkwell Lowry assay).

[0157] Animal Studies Design

[0158] Random-bred, NZW female rabbits weighing approximately 3-3.6 kgwere used in this study. The apolipoprotein-phospholipid complexes wereinjected intravenously by slow bolus infusion into the marginal earvein. For blood sampling, approximately 1-2 ml of blood was collectedfrom the lateral ear vein in the non-injection ear (or in some cases,from the medial artery) using a 3 ml syringe without anticoagulant. Alltubes collected without anticoagulant were kept at room temperature for30-60 min for clotting. Blood samples were centrifuged within 1 hourafter collection and the serum from each sample was separated intoaliquots and stored a −80° C. until time of analysis.

[0159] Three rabbits received r-proApoA-I-POPC complexes while the otherthree received r-proApoA-I-SM complexes. Complexes were administered at15 mg/kg dose based on the protein content. Blood samples were collectedat pre-bleed, 5 min, 30 min, 1 h, 2 h, 24 h, and 48 h.

[0160] Analysis of Blood Samples

[0161] Serum phospholipid (Phospholipid B, Kit # 990-54009, WakoChemicals GmbH, Neuss, Germany), triglycerides (Triglycerides, Kit #1488872, Boehringer Mannheim Corporation, Indianapolis, Ind.), totalcholesterol and unesterified cholesterol were determined withcommercially available kits for a Hitachi 912 Automatic Analyzer (RocheDiagnostics Corporation, Indianapolis, Ind.).

[0162] Lipoprotein profiles were analyzed using gel filtrationchromatography on a Superose 6HR 1×30 cm column equipped with on-linedetection for total or free cholesterol as described by Kieft et al. (JLipid Res 1991; 32:859-866, 1991). Esterified cholesterol in serum andin the lipoprotein fractions VLDL, LDL and HDL was calculated bysubtracting free cholesterol from total cholesterol values.

[0163] Data Analysis

[0164] The time courses of absolute and percent changes in serum free(unesterified) cholesterol, HDL free (unesterified) cholesterol wereplotted. Area under the curve for mobilized cholesterol were calculatedby a trapezoidal method and compared for variousr-pro-ApoA-I-phospholipid complexes.

6.3.2. RESULTS AND DISCUSSION

[0165] ProApoA-I-SM and proApoA-I-POPC complexes (1:2 ratio of proteinweight/phospholipid weight) were administered at 15 mg/kg dose (based onprotein content) to NZW female rabbits. Unexpectedly, a significantlyhigher mobilization of free cholesterol was observed with administrationof proApo-I-SM complexes compared to proApo-I-POPC complexes. The HDLfree cholesterol increased by 31 mg/dl after 30 min followingadministration of proApoA-I-SM versus by 7 mg/dl 30 min followingadministration of proApoA-I-POPC complexes (FIG. 1).

[0166] In addition, large differences were observed in areas under thecurves of free cholesterol and HDL free cholesterol concentration versustime for 0 to 24 h (AUC_(0-24 h)) time period.

[0167] For HDL free cholesterol AUC0-24 h values were 22.3 mg*hr/dl and354.2 mg*hr/dl for r-proApoA-I-POPC and r-proApoA-I-SM complexesrespectively. This indicates that r-proApoA-I-SM complexes mobilized 16times more HDL-free cholesterol in 24 hours compared to r-proApoA-I-POPCcomplexes.

[0168] All cited references are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication, patent or patent application was specificallyand individually indicated to be incorporated herein by reference in itsentirety for all purposes.

[0169] The citation of any publication is for its disclosure prior tothe filing date and should not be construed as an admission that thepresent invention is not entitled to antedate such publication by virtueof prior invention.

[0170] Many modifications and variations of this invention can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. The specific embodiments described are offeredby way of example only, and the invention is to be limited only by theterms of the appended claims along with the full scope of equivalents towhich such claims are entitled.

What is claimed is:
 1. A method of treating dyslipidemia or a diseaseassociated with dyslipidemia, comprising administering to a subject anapolipoprotein-sphingomyelin complex comprising apolipoprotein andsphingomyelin in an amount effective to achieve a serum level of free orcomplexed apolipoprotein in the range of 10 mg/dL to 300 mg/dL above abaseline level before administration.
 2. The method of claim 1 in whichthe disease associated with dyslipidemia is selected from the groupconsisting of coronary heart disease; coronary artery disease;cardiovascular disease, hypertension, restenosis, vascular orperivascular diseases; dyslipidemic disorders; dyslipoproteinemia; highlevels of low density lipoprotein cholesterol; high levels of very lowdensity lipoprotein cholesterol; low levels of high densitylipoproteins; high levels of lipoprotein Lp(a) cholesterol; high levelsof apolipoprotein B; atherosclerosis (including treatment and preventionof atherosclerosis); hyperlipidemia; hypercholesterolemia; familialhypercholesterolemia (FH); familial combined hyperlipidemia (FCH);lipoprotein lipase deficiencies, such as hypertriglyceridemia,hypoalphalipoproteinemia, and hypercholesterolemialipoprotein.
 3. Themethod of claim 1 in which the apolipoprotein is selected from the groupconsisting of preproapoliprotein, preproApoA-I, proApoA-I, ApoA-I,preproApoA-II, proApoA-II, ApoA-II, preproApoA-IV, proApoA-IV, ApoA-IV,ApoA-V, preproApoE, proApoE, ApoE, preproApoA-I_(Milano),proApoA-I_(Milano), ApoA-I_(Milano), preproApoA-I_(Paris),proApoA-I_(Paris), and ApoA-I_(Paris).
 4. The method of claim 3 in whichthe apolipoprotein is a homodimer.
 5. The method of claim 3 in which theapolipoprotein is a heterodimer.
 6. The method of claim 1 in which theratio of SM:apolipoprotein of the complex is in the range of 1:1 to200:1 (mole/mole).
 7. The method of claim 1 in which the ratio ofSM:apolipoprotein of the complex is in the range of 1:2 to 200:1(mole/mole).
 8. The method of claim 1 in which the ratio ofSM:apolipoprotein of the complex is about 2:1 (wt/wt).
 9. The method ofclaim 1 in which the complex-comprises apolipoprotein and sphingomyelin.10. The method of claim 9 in which the complex further includesphosphatidylcholine.
 11. The method of claim 10 in which thephosphatidylcholine is soybean phosphatidylcholine.
 12. The method ofclaim 1 wherein from 50% to70% of the lipid content of the complex issphingomyelin.
 13. The method of claim 1 wherein from 30-50% of thelipid content of the complex is phosphatidylcholine.
 14. The method ofclaim 1 wherein the range of 10 mg/dL to 300 mg/dL is achieved between 5minutes and 1 day after administration.
 15. The method of claim 1 inwhich the apolipoprotein-sphingomyelin complex is administered at aperiodic interval in an amount of about 40 mg to 2 g per person peradministration.
 16. The method of claim 15 in which administration isintravenous administration.
 17. The method of claim 15 in which theperiodic interval is about every 3 to 15 days.
 18. The method of claim15 in which the periodic interval is about every 5 to 10 days.
 19. Themethod of claim 15 in which the periodic interval is about every 10days.
 20. The method of claim 15 in which the periodic interval isseveral times per day, wherein the dose does not reach a daily toxicdose.
 21. The method of claim 15 in which administration is by slowinfusion with a duration of more than one hour.
 22. The method of claim15 in which administration is by rapid infusion of one hour or less. 23.The method of claim 15 in which administration is by a single bolusinjection.
 24. The method of claim 1 in which the complex isco-administered with bile-acid resins, niacin, statins or fibrates. 25.A method of treating a disease associated with dyslipidemia, comprisingadministering to a subject an apolipoprotein-sphingomyelin complex in anamount effective to achieve a circulating plasma concentrations of aHDL-cholesterol fraction between 10% and 1000% of the initialHDL-cholesterol fraction concentration before administration.
 26. Themethod of claim 25 in which the disease associated with dyslipidemia isselected from the group consisting of coronary heart disease; coronaryartery disease; cardiovascular disease, hypertension, restenosis,vascular or perivascular diseases; dyslipidemic disorders;dyslipoproteinemia; high levels of low density lipoprotein cholesterol;high levels of very low density lipoprotein cholesterol; low levels ofhigh density lipoproteins; high levels of lipoprotein Lp(a) cholesterol;high levels of apolipoprotein B; atherosclerosis (including treatmentand prevention of atherosclerosis); hyperlipidemia;hypercholesterolemia; familial hypercholesterolemia (FH); familialcombined hyperlipidemia (FCH); lipoprotein lipase deficiencies, such ashypertriglyceridemia, hypoalphalipoproteinemia, andhypercholesterolemialipoprotein.
 27. The method of claim 25 in which theapolipoprotein is selected from the group consisting ofpreproapoliprotein, preproApoA-I, proApoA-I, ApoA-I, preproApoA-II,proApoA-II, ApoA-II, preproApoA-IV, proApoA-IV, ApoA-IV, ApoA-V,preproApoE, proApoE, ApoE, preproApoA-I_(Milano), proApoA-I_(Milano),ApoA-I_(Milano), preproApoA-I_(Paris), proApoA-I_(Paris), andApoA-I_(Paris).
 28. The method of claim 27 in which the apolipoproteinis a homodimer.
 29. The method of claim 27 in which the apolipoproteinis a heterodimer.
 30. The method of claim 25 in which the ratio ofSM:apolipoprotein of the complex is in the range of 1:1 to 200:1(mole/mole).
 31. The method of claim 25 in which the ratio ofSM:apolipoprotein of the complex is in the range of 1:2 to 200:1(mole/mole).
 32. The method of claim 25 in which the ratio ofSM:apolipoprotein of the complex is about 1:2 (wt/wt).
 33. The method ofclaim 25 wherein the circulating plasma concentration of theHDL-cholesterol fraction is achieved between 5 minutes and 1 day afteradministration.
 34. The method of claim 25 in which the complexcomprises apolipoprotein and sphingomyelin.
 35. The method of claim 34in which the complex further includes phosphatidylcholine.
 36. Themethod of claim 35 in which the phosphatidylcholine is soybeanphosphatidylcholine.
 37. The method of claim 25 wherein from 50% to70%of the lipid content of the complex is sphingomyelin.
 38. The method ofclaim 25 wherein from 30-50% of the lipid content of the complex isphosphatidylcholine.
 39. The method of claim 25 in which theapolipoprotein-sphingomyelin complex is administered at a periodicinterval in an amount of about 40 mg to 2 g per person peradministration.
 40. The method of claim 39 in which administration isintravenous administration.
 41. The method of claim 39 in which theperiodic interval is about every 3 to 15 days.
 42. The method of claim39 in which the periodic interval is about every 5 to 10 days.
 43. Themethod of claim 39 in which the periodic interval is about every 10days.
 44. The method of claim 39 in which the periodic interval isseveral times per day, wherein the dose does not reach a daily toxicdose.
 45. The method of claim 39 in which administration is by slowinfusion with a duration of more than one hour.
 46. The method of claim39 in which administration is by rapid infusion of one hour or less. 47.The method of claim 39 in which administration is by a single bolusinjection.
 48. The method of claim 39 in which the complex isco-administered with bile-acid resins, niacin, statins, or fibrates. 49.A method of treating dyslipidemia or a disease associated withdyslipidemia, comprising administering to a subject anapolipoprotein-sphingomyelin complex in an amount effective to achieve acirculating plasma concentration of a HDL-cholesterol fraction between30 and 300 mg/dL.
 50. A method of treating dyslipidemia or a diseaseassociated with dyslipidemia, comprising administering to a subject anapolipoprotein-sphingomyelin complex in an amount effective to achieve acirculating plasma concentration of cholesteryl esters between 30 and300 mg/dL.
 51. The method of claim 49 wherein the circulating plasmaconcentration of the HDL-cholesterol fraction is achieved between 5minutes and 1 day after administration.
 52. The method of claim 49wherein the circulating plasma concentration of cholesteryl esters isachieved between 5 minutes and 1 day after administration.